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Journal of Pharmaceutical and Biomedical Analysis 144 (2017) 12–24

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

j o ur nal ho me page: www.elsev ier .com/ lo cate / jpba

nalysis of stereoselective drug interactions with serum proteins byigh-performance affinity chromatography: A historical perspective

hao Li, David S. Hage ∗

epartment of Chemistry, University of Nebraska, Lincoln, NE, USA

r t i c l e i n f o

rticle history:eceived 5 December 2016eceived in revised form 6 January 2017ccepted 10 January 2017vailable online 11 January 2017

eywords:

a b s t r a c t

The interactions of drugs with serum proteins are often stereoselective and can affect the distribution,activity, toxicity and rate of excretion of these drugs in the body. A number of approaches based on affinitychromatography, and particularly high-performance affinity chromatography (HPAC), have been used astools to study these interactions. This review describes the general principles of affinity chromatographyand HPAC as related to their use in drug binding studies. The types of serum agents that have beenexamined with these methods are also discussed, including human serum albumin, �1-acid glycoprotein,

igh-performance affinity chromatographyrug-protein bindingtereoselective interactionsuman serum albumin1-Acid glycoproteinipoproteins

and lipoproteins. This is followed by a description of the various formats based on affinity chromatographyand HPAC that have been used to investigate drug interactions with serum proteins and the historicaldevelopment for each of these formats. Specific techniques that are discussed include zonal elution,frontal analysis, and kinetic methods such as those that make use of band-broadening measurements,peak decay analysis, or ultrafast affinity extraction.

© 2017 Elsevier B.V. All rights reserved.

. Introduction

The interactions of drugs with proteins are often stereoselective1–5]. When these interactions involve agents such as serum pro-eins, they can play an important role in the distribution, activity,ate of excretion, and toxicity of drugs in the body [6,7]. Informationn these interactions can be valuable in the design and optimiza-ion of chiral separations [1,3,8–11], as well as in predicting howrugs may behave in the human body, in understanding drug-drug

nteractions, and in determining the optimum dosages that shoulde used with such agents for treating patients [6,7,12–17].

The binding of drugs with serum agents is usually a reversiblerocess and may involve several possible proteins or relatedarriers. Common examples of these binding agents are the pro-eins human serum albumin (HSA) and �1-acid glycoproteinAGP), as well as more complex species such as lipoproteins6,7,12–17]. The extent of these interactions is often significant,ith 43% of 1500 most common drugs having 90% or greater

inding to serum proteins and carrier agents [18]. As a result,btaining data on these processes is an important part of the

∗ Corresponding author at: Chemistry Department, University of Nebraska, Lin-oln, NE, 68588-0304, USA.

E-mail address: dhage1@unl.edu (D.S. Hage).

ttp://dx.doi.org/10.1016/j.jpba.2017.01.026731-7085/© 2017 Elsevier B.V. All rights reserved.

absorption/distribution/metabolism/excretion (ADME) data thatare needed prior to the approval of a new drug in the U.S. [7,18].

There are many techniques that have been utilized to study druginteractions with serum proteins. These methods have includedequilibrium dialysis, ultrafiltration, absorption spectroscopy, fluo-rescence spectroscopy, surface plasmon resonance spectroscopy,X-ray crystallography, and nuclear magnetic resonance spec-troscopy [19–21]. This review will discuss an alternative group oftechniques that are based on affinity chromatography and high-performance affinity chromatography (HPAC) [6–8,22]. The generalprinciples of these methods and the types of serum proteins thatthese methods have been used to examine will first be consid-ered. This will be followed by an overview of the techniques thathave been developed and employed in affinity chromatography andHPAC to study drug-protein interactions, with an emphasis on thehistorical development of these techniques and their use in thestudy of stereoselective binding.

2. General principles of affinity chromatography and HPAC

Affinity chromatography can be defined as a type of liquid chro-matography which uses a biologically-related agent as a stationary

phase for the analysis or separation of samples [23–25]. This type ofstationary phase, which is often referred to as the “affinity ligand”,makes use of the reversible and specific interactions that are oftenpresent in biological systems, such as the interaction of an enzyme

al and

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Z. Li, D.S. Hage / Journal of Pharmaceutic

ith a substrate or the binding of an antibody with its antigen.his same feature often allows an affinity column to selectivelyind a given target compound and makes this a valuable tool forhe separation or detection of specific agents in biological samples23,24].

Affinity chromatography was first used in 1910 for enzymesolation [26], but it was not until 1968 that this techniqueecame a popular method for compound purification [24,27–29].ork from the late 1960s through the early 1980s mainly used

arbohydrate-based supports such as agarose or cellulose forhe immobilization of binding agents and preparation of affin-ty columns [23,27–30]. This approach is still commonly used forurification purposes and will be referred to in this review asraditional, or low-performance, affinity chromatography [27,29].esearch then began in the early 1980s in combining affinity chro-atography with HPLC-grade supports such as silica. The resultingethod is now called high-performance affinity chromatogra-

hy (HPAC), or high-performance liquid affinity chromatography23,29,31,32]. Other materials besides silica particles that are nowsed in HPAC include modified polystyrene supports and mono-

ithic supports based on silica or organic polymers [32–36]. Thebility to use these supports with HPLC systems and detectors, asell as the speed and precision of the resulting methods, has been

mportant in allowing affinity columns to be used in both analyticalpplications and in the study of interactions such as drug-proteininding [6,8,11,13,17,23].

Fig. 1 shows a few formats that can be used in affinity chro-atography and HPAC for the study of drug-protein binding

6,7,11,13,37,38]. Both of these methods make use of a column thatontains an immobilized agent such as a serum protein. A drug orolute is first injected or applied onto this column in the presence of

mobile phase that has the desired pH, ionic strength and solventomposition for the binding study [6,7,11,13,39]. In the methodf zonal elution, a small sample containing one or more solutes ispplied onto the column and the retention or shape of the resultingeaks is examined. In the technique of frontal analysis, a solution ofhe solute is continuously applied to the immobilized binding agents the amount of solute that passes through the column is moni-ored. These and other approaches (e.g., see Sections 4–6, whichlso include kinetic methods) can provide information on suchhings as the binding constants and rate constants for the inter-ction, as well as the number and types of binding sites involvedn this process. After the study has been completed, the boundolute can be eluted and the original buffer applied to regener-te the column for the next experiment. Either traditional affinityhromatography or HPAC can be used in such methods [6,13,39].owever, HPAC offers the advantages of being easier to automatend allowing these measurements to be carried out with greaterpeed plus better precision and with various types of on-line HPLCetectors [6,13,40].

. Serum proteins examined by affinity chromatography

Affinity chromatography and HPAC have been used to studyrug interactions with a variety of serum proteins and serum bind-

ng agents. Many of these studies have dealt with HSA, or thelosely-related protein bovine serum albumin (BSA) [6,7,13,17].his is due to the importance of these proteins, their use as chiraltationary phases, and the availability of various immobilizationethods that can be used to prepare affinity columns that are

ood models for the behavior of these proteins in their native form

6–11,40–43] (Note: related chromatographic studies with albu-

in from other species have also been reported) [44–47]. HSA has concentration in plasma of 35–50 g/L, a molar mass of 66.5 kDa,nd an isoelectric point of 4.7. It consists of a single chain of 585

Biomedical Analysis 144 (2017) 12–24 13

amino acids which is stabilized by 17 internal disulfide bonds [16].This protein has two major binding sites for drugs, which are oftenreferred to as Sudlow sites I and II [16,48]. Sudlow site I, also knownas the warfarin-azapropazone site, is a hydrophobic pocket in sub-domain IIA of HSA. Sudlow site II, or the indole-benzodiazepine site,is located in subdomain IIIA [13,16,48]. There are additional bind-ing sites for some other drugs and solutes on this protein (e.g., thedigitoxin and tamoxifen sites), as well as sites for fatty acids, metalions, and endogenous solutes (e.g., bilirubin) [13,16].

HPAC and affinity chromatography have been used to exam-ine the binding by many chiral and achiral drugs and soluteswith HSA [6–8,13,17,21,49]. Examples of a few chiral drugs thathave been separated and studied in this manner, as is illustratedin Fig. 2(a), include barbiturates, benzodiazepines, benzothiadi-azeines, coumarins (e.g., warfarin), �-arylpropionic acids (e.g.,ibuprofen), tryptophan, and thyronines, among numerous others[6,8,10,11,13,21,37,43,50–59]. This has included both measure-ments of the overall binding by such solutes with HSA, as wellas more detailed studies of the equilibrium constants and num-ber of binding sites for these interactions [6,8,10,11,13,21,37]. Insome cases, the rates of these interactions have also been examined[22], along with the effects of varying parameters such as the tem-perature, pH, ionic strength, or solvent polarity on these processes[7,8,10,13].

AGP is another serum protein that has been the subject of manystudies based on affinity chromatography or HPAC [7,8,13]. Thisprotein has been popular for use as a chiral stationary phase, asshown in Fig. 2(b) [8–10,38,60]. However, it has only been overthe last decade in which suitably mild immobilization schemeshave been developed to place AGP into affinity columns in a formthat closely follows the behavior of this protein in its originalform [7,60–62]. AGP is a heterogeneous glycoprotein with a molarmass of 41 kDa and a normal concentration in human serum of0.5–1.0 mg/mL. This protein consists of a single chain of 183 aminoacids and has an isoelectric point of 2.7–3.9 [63–66]. AGP also con-tains five N-linked glycan chains and has a carbohydrate content of45% (w/w) [67,68]. AGP is believed to have one flexible binding sitefor many basic and neutral drugs, as well as for some acidic drugs[63–66,69–71].

A number of reports based on HPAC have examined the chi-ral separation and binding of drugs on AGP columns [7–10]. Someof these studies have looked at the effects of temperature andmobile phase composition on the retention and chiral separation ofsuch drugs as atenolol, warfarin, verapamil, methadone, atropine,disopyramide, ketamine, and various profens (e.g., ketoprofenand ibuprofen) [8–10,72–75]. In addition, detailed thermodynamicstudies have been carried out with this protein and such drugsas propranolol, carbamazepine, and warfarin [61,62,76–78]. Morerecent studies have also employed HPAC and affinity columns toexamine the kinetics for some of these interactions [79,80].

Plasma lipoproteins such as high-density lipoprotein (HDL),low-density lipoprotein (LDL) and very low-density lipoprotein(VLDL) have also been successfully immobilized in recent years andcharacterized by HPAC for their drug binding properties [81–84].Lipoproteins are soluble complexes of lipids and proteins (i.e.,apolipoproteins) [85–87]. The general structure of lipoproteinsconsists of a non-polar core of triacylglycerol and cholesterol esterswhich is covered with a phospholipid monolayer that also containsapolipoproteins [86,87]. Lipoproteins such as HDL, LDL or VLDLhave normal concentrations in human serum of around 280, 410, or150 mg/dL, respectively [86]. These lipoproteins bind and transporthydrophobic compounds and lipids such as cholesterols and tria-

cylglycerols throughout the body [85,86,88]. However, these carrieragents can also bind and carry various basic or hydrophobic drugsin the bloodstream [7,15,85,86,88].

14 Z. Li, D.S. Hage / Journal of Pharmaceutical and Biomedical Analysis 144 (2017) 12–24

F y of dri

tehafHbwehun

4

cFtopiro[tioe[bHc

ig. 1. General approaches used in affinity chromatography and HPAC for the studn Fig. 2(b) and Fig. 4(a) and are adapted with permission from Refs. [37,38].

Recent work with HPAC and lipoprotein columns have shownhat drugs can interact with these binding agents through sev-ral possible mechanisms, including both partitioning into theydrophobic core and saturable interactions (e.g., with sites onpolipoproteins) [81–83]. Both of these processes have been notedor propranolol with HDL, LDL or VLDL, as well as for verapamil withDL [81–84]. The binding parameters for these interactions haveeen measured by HPAC, and the change in these binding constantsith temperature has been investigated. In the case of LDL, stereos-

lective binding has been observed for R- and S-propranolol, whichas been shown by HPAC to be due to the presence of both sat-rable and non-saturable binding for the R-enantiomer but onlyon-saturable binding for the S-enantiomer [81].

. Zonal elution

The most common method that has been used with affinityolumns to study drug-protein interactions is zonal elution (seeig. 3 and top of Fig. 1) [6–8,51,54,89,90]. This is also the approachhat is typically used for the injection and separation of drugs andther solutes with HPLC columns that contain chiral stationaryhases, such as those based on HSA or AGP [8–11,13]. Work start-

ng in the late 1980s and early 1990s used zonal elution to measureetention factors, plate heights, separation factors and peak res-lution to aid in the separation of chiral solutes by these columns8,13,43,53,89,90]. This was accompanied by studies that examinedhe effects of the mobile phase pH, ionic strength, solvent polar-ty and temperature on the nature of these separations and theverall degree of stereoselectivity that they provided [89,90]. Anxample of such a study for R- and S-warfarin is shown in Fig. 2(a)

37]. Other reports have examined the stereoselective binding ofenzodiazepines or non-steroidal anti-inflammatory drugs withSA [53,56,90,91], the separation of vinca alkaloids on HSA or AGPolumns [92], the stereoselective binding of D/L-tryptophan with

ug-protein binding. The examples shown on the right are described in more detail

HSA [50,93,94], and the interactions of thyronines and thyroxine-related compounds with HSA or BSA [51,52,95].

In the early 1990s, research began in which zonal elution wasemployed to obtain more quantitative information on the inter-actions between drugs and proteins that were used as chiralstationary phases [6–8,11,13]. These efforts built on work begun byDunn and Chaiken in 1974, in which it was shown how zonal elu-tion could be used with low-performance affinity columns to obtainbinding constants for enzyme-inhibitor interactions [96]. One waythis type of experiment can be carried out is by using a column thathas an immobilized protein, onto which is injected a small plug ofa solute or drug in the presence of a mobile phase that containsa known concentration of a competing agent. The retention factorfor the injected solute is then measured as the concentration of thecompeting agent is varied. The change in this retention as a func-tion of the competing agent’s concentration is then fit to variousmodels to see how the injected solute is binding to the protein andthe extent to which this interaction is affected by the competingagent. It is further possible from such a fit to obtain information onthe type of competition and equilibrium constants that are presentfor these solute-protein interactions [6,7,13].

Fig. 3 shows some early experiments that were conducted inthis manner with immobilized HSA in HPLC columns [51,54]. Thechromatograms in Fig. 3(a) show how the retention of an injectedsolute can shift in the presence of a competing agent on this typeof column [51]. Fig. 3(b) illustrates how such data may then beplotted and analyzed to determine the types of interactions thatare present and to estimate some binding parameters [54]. Forinstance, the results obtained in Fig. 3(b) made it possible to deter-mine that R- or S-warfarin and phenylbutazone (i.e., the injectedsolutes) had single-site competition with octanoic acid (i.e., the

competing agent) on the immobilized HSA column. It was furtherpossible from these plots to determine the association equilibriumconstants for octanoic acid at its site of competition with each ofthese solutes [54]. Similar results and related equations have been

Z. Li, D.S. Hage / Journal of Pharmaceutical and Biomedical Analysis 144 (2017) 12–24 15

F n the pa lumn[

up[

ttbw[vcaieftis

ig. 2. Separation of (a) R- and S-warfarin on a high-performance HSA column and ind detection of R- and S-norketamine in plasma using a high-performance AGP co37,38].

sed to examine the interactions of many other drugs and com-ounds with HSA, AGP, lipoproteins, and related binding agents6,7,13,17,50,55,60,82–84,97–102].

A variation of this approach appeared in 1992, when zonal elu-ion competition studies were carried out by using injected soluteshat were probes for particular sites on a protein [52]. This workegan with competition studies such as those shown in Fig. 3(b),hich used R- or S-warfarin as probes for Sudlow site I of HSA

37,50–52,54]. It was then shown that equations like the one pro-ided in Fig. 3(b) could be used to obtain binding constants for theompeting agent at specific regions on a protein by injecting sep-rate probe compounds for each region [6,51,52]. This led to thedentification of other site-selective probes that could be used forither HSA or AGP [50–52,60,76,103–105]. Some applications thatollowed included the development of affinity maps for drugs on

hese proteins [7,22,106,107] and reports that examined how mod-fications of these proteins affected local drug interactions at givenites [22,108–110].

resence of various mobile phase concentrations of 1-propranol, and (b) separation with detection based on mass spectrometry. Adapted with permission from Refs.

The shape of plots like those in Fig. 3(b) also makes it possi-ble to discriminate between direct competition and other types ofinteractions that may occur between two drugs or solutes on animmobilized protein [6,7,22,54]. For example, if this plot is linearwith a positive slope, such a result indicates that direct competitionis occurring between the competing agent and injected solute at asingle type of binding site. A slope of essentially zero indicates thatno competition is present, and a negative slope is a signal that pos-itive allosteric effects are occurring. In addition, negative allostericeffects or multi-site binding can be detected by the presence of anon-linear relationship with a positive slope [6,7,22]. It was shownin 2004 how this same data can be used in a more quantitativemanner to not only identify the type of interaction that is takingplace but to determine the coupling constants and binding parame-ters that are present for this interaction [111,112]. This quantitative

approach has been used to examine the allosteric effects that occurbetween tamoxifen and warfarin as these drugs both bind to HSA[113], the allosteric interactions that occur between warfarin and

16 Z. Li, D.S. Hage / Journal of Pharmaceutical and Biomedical Analysis 144 (2017) 12–24

Fig. 3. Examples of competition studies carried out by zonal elution. The chromatograms in (a) were obtained on an HPLC column containing immobilized HSA by usingR-warfarin as the injected solute and mobile phases that contained L-reverse triiodothyronine (L-rT3) at concentrations (from right-to-left) of 0.0, 0.24, 0.49, 0.97 or 1.90 �M.The plot in (b) shows the effects of adding octanoic acid to the mobile phase on the observed retention for (�) R-warfarin, (♦) S-warfarin, and (�) phenylbutazone on an HPLCcolumn containing immobilized HSA. The terms used within the equation in (b) are defined as follows: k’A is the retention factor for the injected solute, X is the retention fort he moa petits from

Sao

tbsfit[tc

his solute due to sites other than those involved in the competitive binding with tdditive, Vm is the void volume, mL is the moles of sites that are involved in the comolute or mobile phase additive at the site of competition. Adapted with permission

-propranolol on AGP [114], the interactions between ibuprofennd benzodiazepines on HSA [115], and the effect of tolbutamiden the interactions of R-warfarin with HSA [116].

Another approach that appeared in the early-to-mid 1990s waso use HPAC to study a binding site on an immobilized proteiny comparing the retention factors or retention times that wereeen for a series of related compounds [22,51]. Early work in thiseld involved a qualitative evaluation to determine which struc-

ural features were the most important contributors to retention13,53]. This was followed by more detailed studies that comparedhe changes in binding affinities that were seen for the relatedompounds or that examined the forces that created such inter-

bile phase additive (i.e., octanoic acid), [I] is the mobile phase concentration of theion process, and K3 or K2 are the association equilibrium constants for the injected

Refs. [51,54].

actions [51,53,91]. This then led to the creation of quantitativestructure-retention relationships (QSRRs), or models of how a pro-tein’s binding site was interacting with a given class of solutes[22,91,117–119]. QSRRs have since been employed to study thebinding of AGP with antihistamines, beta-adrenolytic drugs, orother pharmaceutical agents [7,13,43,119–121] and the binding ofHSA with benzodiazepines or 2,3-substituted and 2-(4-biphenyl)-3-substituted 3-hydroxypropionic acids [118,122,123].

A related application of zonal elution has been to see how theretention of a set of drugs or solutes is changed as the structureof the immobilized protein is altered [7,22]. This method has beenutilized to see how the modification of HSA at specific amino acids

Z. Li, D.S. Hage / Journal of Pharmaceutical and Biomedical Analysis 144 (2017) 12–24 17

Fig. 4. Frontal analysis (a) chromatograms and (b) plots obtained for R-warfarin by HPAC on an HSA column. The results in (a) were acquired at 4 ◦C and by using solutionst rfarin2 eededd

(drdd

5

afaa

hat contained (from right-to-left) 0.22, 0.33, 0.55, 0.76, 1.10, 1.30 or 1.50 �M R-wa5, 37 or 45 ◦C. The term mLapp represents the moles of applied warfarin that were nrug. Adapted with permission from Ref. [37].

e.g., Tyr-411, Trp-214 or Cys-34) will affect its ability to bind torugs or site-specific probes [124–128]. This same method has beenecently used to see how non-enzymatic glycation, as occurs duringiabetes, affects the ability of HSA to bind to sulfonylureas and otherrugs or solutes [108–110].

. Frontal analysis

Another method that is often used in affinity chromatography

nd HPAC for studying stereoselective drug-protein interactions isrontal analysis, or frontal affinity chromatography [6,7,22]. Thispproach was illustrated in the bottom portion of Fig. 1 [37]. Frontalnalysis was first used with low-performance affinity chromatog-

. The results in (b) were generated at temperatures (from bottom-to-top) of 4, 15, to reach the mean point of the breakthrough curve at a given concentration of this

raphy in 1975 by Kasai and Ishii to examine the interactions oftrypsin and related enzymes with immobilized peptides [129]. Asimilar low-performance approach was used in 1978 and 1979 tostudy the binding of salicylate with BSA [130,131]. This methodwas then used with HPAC starting in 1992 through 1994 to exam-ine the binding of HSA with various solutes (see example in Fig. 4)[37]. These studies originally involved characterization of the stere-oselective interactions of R- or S-warfarin and D- or L-tryptophanwith HSA [37,50]. This method has now become a routine tool for

characterizing the overall interactions and binding sites of variousdrugs and solutes with proteins such as HSA, AGP, and lipoproteins[7,11,13,22].

18 Z. Li, D.S. Hage / Journal of Pharmaceutical and Biomedical Analysis 144 (2017) 12–24

ity chR

aittacfptpaes

tmateiiolhdw

tvo[eobd

Fig. 5. General principles behind frontal affineproduced with permission from Ref. [140].

Fig. 4 provides some typical results from these early frontalnalysis studies that made use of HPAC [37]. This type of exper-ment is usually carried out by continuously applying a solutionhat contains a known concentration of the drug or desired soluteo a column that contained an immobilized binding agent, suchs HSA or AGP. As the solute binds to the immobilized agent, theolumn begins to become saturated and a breakthrough curve isormed as more solute elutes from the column [6,22]. The meanosition of this curve is then determined at several solute concen-rations. If sufficiently fast association and dissociation rates areresent between the applied solute and the immobilized bindinggent, the resulting data can be used to provide information on thequilibrium constants for the interaction and the number of bindingites that are involved in this interaction [6,22].

Early work in this area focused on the examination of systemshat could be described by relatively simple single-site binding

odels and linear fits to the data [37,50]. However, it is nowlso common to employ more complex models and equationshat may involve non-linear fits [7,22]. Examples of such mod-ls include those that involve multi-site binding, non-saturablenteractions, and combinations of saturable plus non-saturablenteractions, such as have been observed for the interactionsf some drugs with modified forms of HSA and with AGP oripoproteins [6,22,76–78,80–84,108–110,132–134]. This approachas further been employed in the study of other systems, such asrug-receptor interactions [135] and the binding of various solutesith lectins or enzymes [136–139].

An important advantage of this method over zonal elution ishat the same experiments can be used in frontal analysis to pro-ide information on both the equilibrium constants and numberf binding sites that are involved in a solute-protein interaction7,22]. In addition, frontal analysis can be used to provide precise

stimates of these values, especially when this technique is carriedut in the form of HPAC [6]. Like zonal elution, frontal analysis cane used with various supports and detection methods [6,7,22]. Oneisadvantage of frontal analysis is that it does tend to take longer

romatography-mass spectrometry (FAC-MS).

to perform than zonal elution, and it requires a larger amount ofsolute because studies must be carried out with many solutions ofthe applied compound [6,22].

Besides providing information on the overall processes involvedin a drug-protein interaction, frontal analysis can be used to studythe competition between two compounds for a protein or bindingagent in an affinity column [22]. This is usually done by com-bining frontal analysis with mass spectrometry, giving a hybridmethod known as frontal affinity chromatography-mass spec-trometry (FAC-MS), as is illustrated in Fig. 5 [6,136,138,140]. Thismethod appeared in the late 1990s through early 2000s and typ-ically uses an applied solution that contains both a solute and acompeting agent [136,140]. The resulting breakthrough curves arethen examined to see how the concentration of the competingagent affects binding by the solute to the column. This data, in turn,makes it possible to see if either direct competition or allostericeffects are present between these applied solutes [6,136,140–144].This method can also be used to compare the binding of a set ofcompeting agents to the immobilized agent and to determine theequilibrium constants for these interactions [136,138,140–144].Recent examples of this approach have included studies of thebinding of drugs with receptor domains [135] and the binding anddisplacement properties of flavonoids in the presence of histonedeacetylase SIRT6 [141]. FAC-MS has also been employed in severalstudies to screen mixtures of drug candidates such as oligosac-charides, enzyme inhibitors and peptides for their binding andinteractions with targets such as lectins, enzymes, receptors, andantibodies [136,140,143–146].

6. Kinetic methods

Zonal elution and frontal analysis experiments usually focus on

equilibria that occur between an injected or applied solute and animmobilized binding agent. However, it is further possible to useaffinity chromatography and HPAC to examine the rate of this inter-action [22,39,147–149]. The earliest approach that was developed

Z. Li, D.S. Hage / Journal of Pharmaceutical and Biomedical Analysis 144 (2017) 12–24 19

Fig. 6. Use of band-broadening measurements to examine the interaction kinetics for the enantiomers of phenytoin metabolites with HSA immobilized onto an HPLC column.The chromatograms in (a) were obtained at flow rates (from bottom-to-top) of 0.25, 0.5, 1.0 or 2.0 mL/min for injections of racemic 5-(3-hydroxyphenyl)-5-phenylhydantoin(i.e., m-HPPH; see structure given within this figure, were the asterisk shows the chiral center). The solid lines in (a) show the results for m-HPPH and the dashed lines showthe results for sodium nitrate (i.e., a non-retained solute). The plot in (b) was obtained for the second eluting enantiomer of 5-(4-hydroxyphenyl)-5-phenylhydantoin (i.e.,p olumns e enand rom R

f[as[mawmd

so

-HPPH). In (b), HR is the total plate height measured for an enantiomer on the HSA colute) on a control column; u is the linear velocity, k is the retention factor for thissociation rate constant for the enantiomer with HSA. Adapted with permission f

or this purpose is one based on band-broadening measurements39,147–149]. This approach was suggested as early as 1975 [150]nd was used with low-performance affinity columns to study theelf-association of neurophysin II and its binding to a neuropeptide151]. This method was then explored for use with HPAC in the

id-1980s for the study of lectin-sugar interactions [39,152,153]nd was adapted for use in the study of drug or solute interactionsith HSA by the mid-1990s [93,154]. Band-broadening measure-ents have since been used in various forms to study the binding of

rugs and drug metabolites with both HSA and AGP [80,155–157].

A band-broadening experiment is usually carried out by mea-

uring the plate heights for a drug or solute at one or more flow ratesn an affinity column and then comparing these results to those that

, and HM is the plate height determined for the same enantiomer (or a non-retainedtiomer, Hk is the plate height due to stationary phase mass transfer, and kd is the

ef. [157].

are obtained on an inert control column [147–149]. This compari-son makes it possible to find the part of the total plate height for thedrug or solute that is a result of stationary phase mass transfer (Hk).The plate height term that is obtained for this process is related tothe dissociation rate for the drug/solute as it is released from theimmobilized agent and can be used, through various approaches,to obtain the dissociation rate constant [39,147–149]. An exampleof such a study is provided in Fig. 6 [157].

Several variations on this band-broadening approach have beenreported. One form is a technique known as the plate height method

[22,39,147–149]. In this method, a plot is made of Hk versus theinjection flow rate or linear velocity, or a combined function ofthe flow rate/linear velocity and the retention factor for the solute.

20 Z. Li, D.S. Hage / Journal of Pharmaceutical and

Fig. 7. Scheme for (a) the injection of a drug/protein sample onto an HSA microcol-umn, and (b) use of ultrafast affinity extraction with this sample and microcolumn todetermine the association equilibrium constant (Ka) or global affinity (nKa’) for thedrug-protein interaction in solution (middle panel) or the dissociation rate constant(

R

Tr[t[paiar[da

c[cfirablrdtawdmd

fteettq

environment and their chirality, Chem. Soc. Rev. 39 (2010) 4466–4503.

kd) for the drug from the soluble drug-protein complex (bottom panel).

eproduced with permission from Ref. [162].

he slope of this plot can then be used to provide the dissociationate constant for the solute with the immobilized binding agent22,147–149]. The stereoselective interactions of HSA with D/L-ryptophan and R/S-warfarin have been examined by this approach93,154]. Another form of the band-broadening approach is peakrofiling, which makes use of the difference in plate heights thatre measured for a drug or solute on an affinity column and on annert control column (see Fig. 6) [157]. This difference is determinedt one or more flow rates and is used to calculate the dissociationate constant for the solute with the immobilized binding agent155]. Peak profiling has been used in recent years to estimate theissociation rate constants for a number of chiral or achiral drugsnd solutes with both HSA and AGP [80,155–158].

Another method that can be used with HPAC and affinityolumns to obtain rate constants is the peak decay method22,147–149]. The peak decay method makes use of small affinityolumns and elution conditions in which the solute is preventedrom rebinding to the immobilized agent as the retained solutes released from the column [159–161]. This method was firsteported in the mid-1980s and used displacing agents to dissoci-te solutes (e.g., fluorescent sugars) that had moderate-to-stronginding to an immobilized agent in the column (e.g., an immobi-

ized lectin) [39,159]. When this was done at a suitably high flowate, the elution profile of the solute was found to form a first-orderecay curve with a slope that gave the dissociation rate constant forhe solute from the column [22,39,159]. An alternative peak decaypproach was described in 2009 that could be used for systems witheak-to-moderate strength interactions and that did not require aisplacing agent [160]. This second method has been employed toeasure dissociation rate constants for various chiral and achiral

rugs with both HSA [79,160,161] and AGP [79].An alternative approach that can be used with affinity columns

or either equilibrium or kinetic studies is ultrafast affinity extrac-ion (see Fig. 7) [149,162–169]. This method was first used forquilibrium-related measurements in 2001 [166] and for bothquilibrium and rate constant measurements in 2014 [162]. This

echnique can be used to study a drug-protein interaction in solu-ion by using a secondary binding agent in a small affinity column touickly extract the non-bound form of the solute from an injected

Biomedical Analysis 144 (2017) 12–24

sample. This extracted fraction is then measured and used to pro-vide information on both the rate and extent of the drug-proteinbinding. Both antibodies and more general binding agents such asHSA have been used to extract the non-bound form of drugs in thismethod [162–169]. The column size and flow are selected to mini-mize or control the level of dissociation that occurs by the solutesfrom soluble proteins as the sample passes through the column;these conditions often involve the use of sample residence times inthe affinity column that range from a few hundred milliseconds upto a few seconds [149].

Systems with a wide range of binding strengths and dissoci-ation rates have been examined by ultrafast affinity extraction[149,165,167]. Initial work with this method used it with smallantibody columns to measure the non-bound fraction of warfarinin samples that contained this drug and HSA [166] and the non-bound fractions of phenytoin and thyroxine in serum [165,167].This method was then adapted for use with small HSA columnsto estimate the equilibrium constants for soluble HSA with war-farin, ibuprofen and imipramine [163] and to measure both thenon-bound fractions and binding constants for R- and S-warfarinin serum and in mixtures with HSA [169]. This method was thenadapted for kinetic studies and used with small HSA columns tostudy the interactions of several drugs with HSA [162], along withthe interactions of testosterone with both HSA and sex hormonebinding globulin [168].

7. Conclusions

This review examined the use of affinity chromatography andHPAC for the study of stereoselective drug interactions with serumbinding agents, within an emphasis on the history of this field.The general principles of affinity chromatography and HPAC werefirst discussed, as related to drug-protein binding studies. Vari-ous types of serum proteins and binding agents that have beeninvestigated by these methods were also examined. Several for-mats by which affinity chromatography and HPAC can be used indrug-protein binding studies were then described. These formatsincluded zonal elution and frontal analysis methods, as well as sev-eral approaches for kinetic studies. It was shown how this groupof methods could be used to examine and measure such thingsas the binding constants, rate constants, and number of bindingsites for drugs with agents such as HSA, AGP, and lipoproteins.The use of these approaches in studying drug-drug competition,in characterizing binding sites, and in examining the mechanism ofa drug-protein interaction was also discussed. In addition, severalexamples of these methods were provided. As HPAC and affin-ity chromatography continue to be developed for such work, itis expected that even more applications and formats will appearfor these techniques in the study of stereoselective interactions bydrugs with serum proteins and other binding agents.

Acknowledgements

This work was supported, in part, by the National Institutes ofHealth under grants R01 GM044931 and R01 DK069629, and theNational Science Foundation (NSF) under grant CHE 1309806.

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